Cell Death and Differentiation (2008) 15, 1745–1751 & 2008 Macmillan Publishers Limited All rights reserved 1350-9047/08 $32.00 www.nature.com/cdd Zinc chelation induces rapid depletion of the X-linked inhibitor of apoptosis and sensitizes prostate cancer cells to TRAIL-mediated apoptosis

P Makhov1,2, K Golovine1,2, RG Uzzo1, J Rothman1, PL Crispen1, T Shaw1, BJ Scoll1 and VM Kolenko*,1

The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane- permeable zinc chelator, N,N,N0,N0,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP. Cell Death and Differentiation (2008) 15, 1745–1751; doi:10.1038/cdd.2008.106; published online 11 July 2008

Inhibitor of apoptosis proteins (IAPs) are a family of caspase and selective depletion of XIAP at the post-translational level. inhibitors that selectively bind and inhibit caspases at both the XIAP depletion coincides with increased activation of initiation phase and the execution phase of apoptosis.1 All caspases-3 and -9 and sensitization of PC-3 cells to apoptosis IAPs contain 1–3 baculoviral IAP repeat (BIR) motifs. Each in response to subsequent treatment with TRAIL. BIR domain folds into a functionally independent structure that binds a zinc ion. Additionally, many IAPs contain another zinc- binding motif, the RING domain, which has E3 ubiquitin ligase Results activity.2 Of all the members of the IAP family, XIAP has received the Chelation of intracellular zinc induces rapid depletion of most attention, possibly because it is the only member of this XIAP. The IAP family members suppress cell death by family that is able to directly inhibit both the initiation and inhibiting caspase activity. The zinc-binding BIR domains are execution phases of the caspase cascade.3 The BIR2 domain responsible for this inhibitory function. Therefore, we of XIAP binds and inhibits caspases-3 and -7. Overexpression examined the impact of intracellular zinc chelation on the of cDNA corresponding to the BIR2 domain inhibits apoptosis expression of the IAP family members. As seen in Figure 1a, from both death receptor and mitochondrial pathway stimuli, treatment with TPEN induces a rapid decrease in XIAP consistent with its ability to inhibit effector caspases.4,5 The protein levels in human PC-3 prostate cancer cells. Our BIR3 domain of XIAP binds and inhibits caspase-9, an apical experiments show that the inhibition of XIAP expression is caspase in the mitochondrial arm of the apoptotic pathway.5,6 selective, as TPEN had no effect on the levels of other zinc- Overexpression of cDNA corresponding to the BIR3 domain containing members of the IAP family, namely cIAP1, inhibits apoptosis in response to stimuli of the mitochondrial cIAP2 and survivin (Figure 1a). Notably, a decrease in pathway of caspase activation, such as Bax, but not stimuli of XIAP protein level was detectable at an extracellular TPEN the death receptor pathway.4 XIAP levels are elevated in concentration as low as 6 mM and was dose-dependent many cancer cell lines and suppression of XIAP protein levels (Figure 1b). sensitizes cancer cells to chemotherapeutic agents.3 To validate our observation that zinc chelation induces a Here, we demonstrate that treatment of PC-3 prostate rapid decrease of XIAP, we constructed an expression vector cancer cells with the zinc-specific chelator N,N,N0,N0,-tetra- encoding a doubly tagged XIAP modified to incorporate both kis(2-pyridylmethyl) ethylenediamine (TPEN) induces rapid an amino-terminal HA tag and a carboxy-terminal FLAG tag in

1Department of Surgical Oncology, Division of Urology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA *Corresponding author: VM Kolenko, Department of Surgical Oncology, Division of Urology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. Tel: 215 728 5620; Fax: 215 728-4333; E-mail: [email protected] 2These authors contributed equally to this work Keywords: XIAP; zinc; cancer; prostate; apoptosis Abbreviations: BIR, baculoviral IAP repeat; PARP, poly(ADP-ribose) polymerase; PSA, prostate specific antigen; RING, really interesting new gene; TPEN, N,N,N 0,N 0,-tetrakis(2-pyridylmethyl) ethylenediamine; TRAIL, TNF-related apoptosis inducing ligand; XIAP, X-linked inhibitor of apoptosis protein Received 20.3.08; revised 16.6.08; accepted 16.6.08; Edited by DL Vaux; published online 11.7.08 Zinc chelation induces depletion of XIAP P Makhov et al 1746

Figure 1 Zinc chelation induces rapid depletion of XIAP in PC-3 cells. (a) Cells were cultured for the indicated periods of time with TPEN (8 mM). The cell lysates were subjected to SDS-PAGE, blotted and probed with specific antibodies. (b) Cells were cultured in medium alone or treated with indicated concentrations of TPEN for 2 h. The cell lysates were subjected to SDS-PAGE followed by western blotting with anti-XIAP antibody. (c) Doubly tagged XIAP containing an amino-terminal HA tag and a carboxy- terminal FLAG tag was expressed in PC-3 cells. TPEN (8 mM) was added to the media for 2 h. XIAP was detected by immunoblotting using anti-FLAG or anti-HA antibodies. (d)[35S]Methionine pulse-chase analysis of XIAP half-life in the presence or absence of TPEN. TPEN (8 mM) was added to the media for the indicated periods of time. Pulse- chase analysis was performed as described in Materials and Methods. (e) XIAP mRNA levels were not decreased in TPEN-treated PC-3 cells. TPEN (8 mM) was added to the media for 2 h. XIAP mRNA levels were detected by RT-PCR using specific primers. (f) The addition of zinc restores XIAP expression after TPEN-mediated depletion. PC-3 cells were pre-incubated with TPEN (8 mM) for 2 h followed by the addition of an equimolar concentration of ZnSO4 to the cell culture medium for 4 h

the same protein. PC-3 cells were transfected with the metals, completely blocked TPEN-induced XIAP degradation. construct followed by incubation with or without TPEN for Although TPEN has been used as a zinc-specific chelator in 2 h. As demonstrated in Figure 1c, the presence of tagged multiple studies,7–9 it has a much higher affinity for copper À20 À16 10 XIAP protein was detectable only in untreated cells. To (Kd ¼ 3 Â 10 M) than for zinc (Kd ¼ 2.6 Â 10 M). This discriminate whether TPEN promotes XIAP degradation or suggests that copper effectively competes with zinc for prevents its synthesis, we examined the half-life of XIAP binding to TPEN and, therefore, ‘neutralizes’ its activity with protein using [35S]methionine pulse-chase analysis. Results respect to XIAP downregulation. In RPMI medium supple- of these experiments clearly demonstrate that exposure of mented with 10% FBS, copper and zinc levels are 0.25 and PC-3 cells to TPEN markedly decreases the half-life of XIAP 4.2 mM, respectively.11 The results of our experiments protein (Figure 1d). To test the possibility that TPEN might demonstrating that a noticeable effect upon XIAP expression induce downregulation of XIAP at the mRNA level, we was detected only when TPEN was used at concentrations examined the levels of XIAP mRNA in PC-3 cells incubated greater than 4 mM (Figure 1b) support the concept that TPEN- with or without TPEN. As shown in Figure 1e, XIAP mRNA mediated reduction of XIAP expression was indeed mediated levels were not decreased in TPEN-treated cells. Therefore, primarily by chelation of zinc and not other physiologically zinc chelation induces depletion of XIAP specifically at the relevant metals. protein level. To determine whether downregulation of XIAP Next, we tested the ability of TPEN to modulate XIAP expression is a reversible process, we added equimolar expression in non-prostate cell lines. Figure 2b demonstrates concentration of ZnSO4 to the cells pretreated with TPEN. The that incubation with TPEN decreased XIAP levels in all tested results presented in Figure 1f demonstrate that the addition of cell lines, including cervical, colon, ovarian, leukemia and zinc restores XIAP expression in TPEN-treated cells. breast cancer cells. To investigate whether the TPEN-induced XIAP down- regulation was mediated specifically via zinc depletion, PC-3 Degradation of XIAP in TPEN-treated cells occurs via cells were exposed to TPEN (8 mM) with the addition of proteasome- and caspase-independent pathways and is equimolar concentrations of physiologically relevant metal prevented by the serine protease inhibitor Pefabloc. A ions (Figure 2a). Addition of zinc and copper, but not other potential mechanism explaining the TPEN-induced decrease

Cell Death and Differentiation Zinc chelation induces depletion of XIAP P Makhov et al 1747 of XIAP at the protein level without a reduction in Figure 3a show that MG-132 did not block the ability of TPEN XIAP mRNA may be its increased degradation by the to reduce the XIAP protein levels, although MG-132 ubiquitin–proteasome or caspase-mediated pathways.12,13 completely prevented TNF-a-induced degradation of IkBa. To address this hypothesis, we pre-incubated PC-3 cells with We have reported previously that caspases-3, -8 and -9 were the proteasome inhibitor MG-132. The results presented in rapidly activated in T lymphocytes treated with TPEN.9 For this reason, we analyzed whether inhibition of caspase activity in TPEN-treated cells prevents XIAP degradation. The inhibition of caspase activity with the pan-caspase inhibitor Z-VAD-FMK failed to prevent TPEN-mediated degradation of XIAP, although the addition of Z-VAD-FMK completely blocked cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP) in PC-3 cells simultaneously treated with TRAIL and methylseleninic acid (Figure 3b). XIAP is a bona fide substrate of Omi/HtrA2 protease, which mediates XIAP cleavage under various stress conditions.14,15 With this fact in mind, we treated cells with Omi’s specific inhibitor, Ucf-101.14 This also failed to prevent XIAP depletion (Figure 3c). Similar results were obtained with ALLM, an effective calpain and cathepsin inhibitor;16 chloroquine, a lysosomal protease inhibitor;17 and CA-074 Me, a cathepsin B inhibitor18 (Figure 3d). To examine the potential contribution of other classes of proteases in TPEN-mediated XIAP degradation, PC-3 cells were pre-incubated with cell-permeable inhibitors specific for Figure 2 (a) Addition of equimolar concentrations of zinc and copper completely serine (Pefabloc), cysteine (E64d) and aspartic (pepstatin A) blocks TPEN-induced XIAP degradation. PC-3 cells were pre-incubated with 8 mM proteases. As demonstrated in Figure 3e, only the serine of KCl, NaCl, CaCl2, MgSO4, ZnSO4, CuSO4 and FeCl2 for 1 h, and then incubated protease inhibitor Pefabloc was able to prevent XIAP with an equimolar concentration of TPEN for 2 h. XIAP expression was detected by depletion in TPEN-treated cells. immunoblotting using an anti-XIAP antibody. (b) TPEN reduces XIAP expression in tumor cell lines of various origins. PC-3 (prostate cancer), HeLa (cervical cancer), HCT116 (colon cancer), A1847 (ovarian cancer), Jurkat (leukemia) and MCF-7 XIAP domains possess variable sensitivity to (breast cancer) cells were cultured in the presence of TPEN (8 mM) for 2 h. XIAP intracellular zinc chelation. The potential sensitivity of was detected by immunoblotting using an anti-XIAP antibody various domains of XIAP to TPEN-mediated degradation was

Figure 3 TPEN-mediated XIAP depletion is prevented by the serine protease-specific inhibitor Pefabloc. (a) PC-3 cells were pre-incubated with 10 mM of the proteasome inhibitor MG-132 for 30 min, followed by incubation with either 8 mM of TPEN for 2 h or 20 ng/ml of TNF-a for 15 min. (b) PC-3 cells were pre-incubated with 50 mM of the pan- caspase inhibitor Z-VAD-FMK for 30 min, followed by incubation with either 8 mM of TPEN for 2 h or 200 ng/ml of TRAIL and 5 mM methylseleninic acid (MSA) for 4 h. PC-3 cells were pre-incubated with (c) Ucf-101 (100 mM); (d) chloroquine (20 mM), CA-074 Me (20 mM) or ALLM (10 mM); (e) Pefabloc (200 mM), E64d (200 mM) or pepstatin A (200 mM) for 1 h, followed by incubation with 8 mM of TPEN for 2 h. Expression of XIAP, IkBa, PARP and actin was detected by immunoblotting with specific antibodies

Cell Death and Differentiation Zinc chelation induces depletion of XIAP P Makhov et al 1748

Figure 4 XIAP domains possess variable sensitivity to intracellular zinc chelation. The indicated XIAP mutants containing the HA tag were expressed in PC-3 cells. Cells were treated with TPEN (8 mM) for 2 h. Expression of XIAP and actin was detected by immunoblotting with anti-HA or anti-actin antibodies, respectively

evaluated by testing the ability of truncated versions of the protein to undergo the degradation after the addition of TPEN to cells transfected with appropriate constructs containing an amino-terminal HA tag. As shown in Figure 4, the amino- terminal portion of XIAP containing the three BIR domains and lacking the RING finger domain was degraded in the presence of TPEN, as were BIR1–2 and the full-length XIAP constructs. In addition, a truncation mutant lacking all BIR domains and containing the RING finger domain (DBIR) was also capable of undergoing the TPEN-induced degradation. In contrast, the BIR2–3 protein was resistant to TPEN-mediated cleavage. These results indicate that the BIR2–3 construct likely lacks the specific motif required for XIAP cleavage.

TPEN promotes activation of caspases-3 and -9 and sensitizes prostate cancer cells to TRAIL-mediated apoptosis. XIAP is probably the only bona fide inhibitor that selectively binds and inhibits caspases-3, -7 and -9.1 With this understanding, we examined whether TPEN- mediated downregulation of XIAP coincides with increased proteolytic processing via caspases-3 and -9 and cleavage of intracellular caspase-3 substrate PARP in cells treated with Figure 5 TPEN-mediated XIAP depletion coincides with increased activation of TRAIL. When PC-3 cells were treated with TPEN alone, the caspases-3 and -9 and sensitization of tumor cells to TRAIL-mediated apoptosis. level of proteolytic activation of caspases-3 and -9 remained (a) PC-3 cells were treated with TPEN (8 mM), TRAIL (200 ng/ml) or the TPEN/ consistently low throughout a 3 h incubation (Figure 5a). TRAIL combination for 3 h. Cell lysates were subjected to SDS-PAGE, blotted and probed with antibodies to full length and cleaved caspases-3 and -9, and PARP. Although treatment of PC-3 cells with TRAIL alone was (b) Caspase-3-like activity in PC-3 cells treated with TPEN (8 mM), TRAIL (200 ng/ capable of inducing noticeable levels of caspases-3 and -9 ml) or the TPEN/TRAIL combination for 3 h. Caspase-3-like activity was examined processing, PARP cleavage was undetectable in cells using the fluorogenic substrate DEVD-AMC, as described in Materials and Methods. treated with TRAIL alone (Figure 5a). Importantly, in Results are mean±S.E.M. (n ¼ 3). (c) Zinc chelation sensitizes prostate cancer mammals, XIAP binds exclusively to activated, processed cells to TRAIL-mediated apoptosis. PC-3 cells were cultured in medium alone or in forms of caspases-3, -7 and -9 and does not interact with the presence of TPEN (8 mM) with or without TRAIL (200 ng/ml) for 4.5 h. The their zymogenic forms.19,20 These findings can explain the percentage of apoptotic cells was determined by the APO-BRDU assay followed by flow cytometry analysis. Columns, means of four different experiments; bars, S.E.M. lack of PARP cleavage in the presence of processed caspases-3 and -9 in cells treated with TRAIL alone. In contrast, when PC-3 cells were treated with TPEN and deficiency contributes to increased activity of caspase TRAIL simultaneously, a clear effect on the processing of family members in cells treated with TRAIL, caspase-3-like caspases-3 and -9 and PARP cleavage was detected activity was examined in cell lysates from PC-3 cells treated (Figure 5a). To confirm that TPEN-mediated XIAP with various combinations of TPEN and TRAIL using the

Cell Death and Differentiation Zinc chelation induces depletion of XIAP P Makhov et al 1749

fluorogenic substrate DEVD-7-amino-4-methyl-coumarin (AMC). As expected, treatment with either TPEN or TRAIL alone failed to induce significant levels of caspase-3-like activity, whereas such an activity was easily detectable in PC-3 cells simultaneously treated with both TPEN and TRAIL (Figure 5b). Given that suppression of XIAP activity/expression sensi- tizes cells to cytotoxic agents,3,21 the pro-apoptotic effect of TPEN in combination with TRAIL was evaluated in PC-3 cells. Treatment with TRAIL alone had no significant effect on cell death in PC-3 cells, as prostate cancer cells are generally resistant to TRAIL-mediated killing. Treatment with TPEN alone also did not induce a significant degree of apoptosis. However, concomitant treatment with TRAIL and TPEN resulted in high levels of DNA fragmentation as early as 4.5 h after treatment initiation (Figure 5c). Furthermore, as demonstrated in Figure 6, direct small interfering RNA- mediated knockdown of XIAP resulted in the upregulation of caspase-3-like activity and TRAIL sensitization, suggesting that knockdown of XIAP by itself was sufficient to reverse resistance to TRAIL-mediated apoptosis in PC-3 cells. Yet, the results of our experiments presented in Figure 6c demonstrate that the level of apoptosis induced by TRAIL in the presence of TPEN was higher than the level of apoptosis induced by TRAIL in cells with XIAP knockdown. This may be due to the ability of TPEN to trigger more profound XIAP depletion compared with shRNA or due to a potential effect of TPEN on other critical components of the apoptotic pathway. The present study provides the first evidence that intracel- lular zinc chelation induces rapid depletion of XIAP in tumor cells of various origins at the post-translational level. Figure 7 schematically illustrates the potential mechanism of tumor cell sensitization to TRAIL via TPEN-induced downregulation of XIAP. Figure 6 Small interfering RNA-mediated knockdown of XIAP sensitizes PC-3 cells to TRAIL-mediated apoptosis. (a) XIAP protein levels in native PC-3 cells and Discussion cells transfected with shRNA targeting XIAP. PC-3 cells were transfected with XIAP shRNA plasmid for 48 h, as described in Materials and Methods. XIAP, cIAP1, The therapeutic potential of XIAP suppression in cancer cIAP2 and actin levels were detected by immunoblotting using the appropriate treatment has encouraged research to identify and antibodies. (b) Knockdown of XIAP causes upregulation of caspase-3-like activity in characterize mechanisms that regulate XIAP expression as PC-3 cells treated with TRAIL. Cells were transfected with XIAP shRNA plasmid, as well as antagonists that block XIAP function. The inhibition or described above, followed by stimulation with TRAIL (200 ng/ml) for 6 h. Caspase-3- like activity was examined using the fluorogenic substrate DEVD-AMC, as described downregulation of XIAP in cancer cells lowers the apoptotic in Materials and Methods. Results are mean±S.E.M. (n ¼ 3). (c) Knockdown of threshold, thereby inducing cell death and/or enhancing the XIAP sensitizes PC-3 cells to TRAIL-mediated apoptosis. Cells were transfected cytotoxic action of chemotherapeutic agents. Indeed, XIAP with XIAP shRNA plasmid, as described above, followed by stimulation with TRAIL antagonist 1396-34 sensitized PC-3 and DU-145 prostate (200 ng/ml), TPEN (8 mM) or a combination of both for 12 h. The percentage of cancer cells to chemotherapeutic agents and TRAIL.22 apoptotic cells was determined by the APO-BRDU assay followed by flow cytometry Moreover, XIAP antisense enhanced pro-apoptotic TRAIL analysis. Columns, means of three different experiments; bars, S.E.M. potency by 12- to 13-fold in DU-145 prostate cancer cells23 demonstrating that knockdown of XIAP itself was sufficient to reverse TRAIL resistance. XIAP. Nevertheless, the exact mechanism underlying the Zinc, in addition to its catalytic role in more than 300 zinc depletion of XIAP under zinc-deficient conditions remains to metalloenzymes, is a known inhibitor of enzymes in general. be elucidated. A possibility also exists that the TPEN- Removal of zinc from an inhibitory zinc-specific enzymatic site mediated pro-apoptotic effect is not exclusively due to XIAP results in a marked increase of enzyme activity.24 For depletion. Further studies are needed to fully explore the example, human tissue kallikreins, a subgroup of extracellular potential mechanisms of action of zinc-chelating agents. serine proteases, including prostate-specific antigen, are Zinc is not the only metal ion influencing XIAP function. inactivated by the reversible binding of zinc (reviewed in the Elevated copper levels result in a conformational change in report by Pampalakis and Sotiropoulou).25 Such findings XIAP, which accelerates its degradation and significantly provide a rationale for our observation that the addition of zinc decreases its ability to inhibit caspase-3 in HEK 293 cells.26 In chelators triggers serine protease-dependent depletion of contrast to our observation that zinc chelation induces specific

Cell Death and Differentiation Zinc chelation induces depletion of XIAP P Makhov et al 1750

Figure 7 A proposed model for the enhanced susceptibility of prostate cancer cells to TRAIL-mediated apoptosis due to zinc chelation. The zinc-specific chelator TPEN induces rapid depletion of XIAP at the post-translational level, abrogating its apoptosis-inhibitory function, and thereby promoting amplification of an apoptotic signaling via activation of caspases-3 and -9

degradation of XIAP (Figure 1a), studies by Mufti et al. show treatment with the inhibitor Gleevec.3,29 Therefore, agents that intracellular copper accumulation also affects other IAPs, capable of completely depleting XIAP in malignant cells might such as Op-IAP and cIAP2. Mufti et al.26 have also reported be superior to small-molecule XIAP inhibitors. Taken that copper induced no significant changes in XIAP mRNA together, our findings indicate that zinc-chelating molecules expression. This finding is in agreement with our data that capable of reducing XIAP expression, in combination with XIAP mRNA levels were not decreased in TPEN-treated cells other treatment modalities, may be beneficial in treating (Figure 1e), indicating that both copper and TPEN can apoptosis resistant tumors. modulate XIAP expression at the post-transcriptional level. Furthermore, it has been suggested that direct binding of copper to cysteine residues within XIAP induces a conforma- Materials and Methods tional change that is associated with decreased stability.26 An Cells and materials. Cell lines were obtained from ATCC (Rockville, MD, intriguing possibility exists that, in addition to direct binding of USA) and maintained in RPMI 1640 medium (Bio-Whittaker, Walkersville, copper to XIAP, it can also substitute for zinc, and thereby MD, USA) supplemented with 10% FCS (Hyclone, Logan, UT, USA), gentamicin promote the instability of XIAP. Indeed, the ability of various (50 mg/l), sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM). metals, including copper and cadmium, to substitute zinc in 27,28 zinc finger domains is well established. Antibodies and reagents. Antibodies to cIAP2, survivin, PARP, HA tag and Recent studies suggest that the presence or development IkBa were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). of resistance may ultimately limit the use of small-molecule Antibodies to cIAP1, XIAP, caspase-3 and caspase-9 were obtained from Cell XIAP inhibitors.3 It is unknown whether malignant cells Signaling Technology (Beverly, MA, USA). Ucf-101, chloroquine, CA-074 ME and acquire mutations in the BIR domains of XIAP and whether ALLM were purchased from Calbiochem (San Diego, CA, USA). Antibodies to FLAG tag, actin, TPEN and TNF-a were obtained from Sigma (St. Louis, MO, USA). these mutations affect XIAP’s function or its response to Z-VAD-FMK, TRAIL, CD95 antibody, DEVD-AMC and MG132 were obtained from inhibitors. It is also unknown whether mutations in XIAP can Biomol (Plymouth Meeting, PA, USA). TNF-a was obtained from Sigma. pSM2 develop after treatment with small-molecule XIAP inhibitors vector encoding XIAP shRNAmir was obtained from Open Biosystems (Huntsville, similar to the development of mutations in ABL kinase after AL, USA).

Cell Death and Differentiation Zinc chelation induces depletion of XIAP P Makhov et al 1751

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Apoptosis 2001; 6: gta atcAGACATAAAAATTTTTTGCTT-30 (reverse) specific primers (restriction sites 419–429. underlined) using pEBB-HA-XIAP vector30 as a template with subsequent cloning 10. Armstrong C, Leong W, Lees GJ. Comparative effects of metal chelating agents on the into BamH1/Cla1 sites of the pEBB-HA plasmid. The pEBB-HA-XIAP vector and neuronal cytotoxicity induced by copper (Cu+2), iron (Fe+3) and zinc in the hippocampus. XIAP truncation mutants were a kind gift from Dr. CS Duckett (University of Brain Res 2001; 892: 51–62. Michigan Medical School, Ann Arbor, MI, USA). Transfections were performed 11. Glassman AB, Rydzewski RS, Bennett CE. Trace metal levels in commercially prepared 12 using TransIT-Prostate transfection kit (Mirus Bio, Madison, WI, USA). tissue culture media. Tissue Cell 1980; : 613–617. 12. Dan HC, Sun M, Kaneko S, Feldman RI, Nicosia SV, Wang HG et al. Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). 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Cell Death and Differentiation